Immuno-PET Imaging of CD69 Visualizes T-Cell Activation and Predicts Survival Following Immunotherapy in Murine Glioblastoma

Glioblastoma (GBM) is the most common and malignant primary brain tumor in adults. Immunotherapy may be promising for the treatment of some patients with GBM; however, there is a need for noninvasive neuroimaging techniques to predict immunotherapeutic responses. The effectiveness of most immunotherapeutic strategies requires T-cell activation. Therefore, we aimed to evaluate an early marker of T-cell activation, CD69, for its use as an imaging biomarker of response to immunotherapy for GBM. Herein, we performed CD69 immunostaining on human and mouse T cells following in vitro activation and post immune checkpoint inhibitors (ICI) in an orthotopic syngeneic mouse glioma model. CD69 expression on tumor-infiltrating leukocytes was assessed using single-cell RNA sequencing (scRNA-seq) data from patients with recurrent GBM receiving ICI. Radiolabeled CD69 Ab PET/CT imaging (CD69 immuno-PET) was performed on GBM-bearing mice longitudinally to quantify CD69 and its association with survival following immunotherapy. We show CD69 expression is upregulated upon T-cell activation and on tumor-infiltrating lymphocytes (TIL) in response to immunotherapy. Similarly, scRNA-seq data demonstrated elevated CD69 on TILs from patients with ICI-treated recurrent GBM as compared with TILs from control cohorts. CD69 immuno-PET studies showed a significantly higher tracer uptake in the tumors of ICI-treated mice compared with controls. Importantly, we observed a positive correlation between survival and CD69 immuno-PET signals in immunotherapy-treated animals and established a trajectory of T-cell activation by virtue of CD69-immuno-PET measurements. Our study supports the potential use of CD69 immuno-PET as an immunotherapy response assessment imaging tool for patients with GBM. Significance: Immunotherapy may hold promise for the treatment of some patients with GBM. There is a need to assess therapy responsiveness to allow the continuation of effective treatment in responders and to avoid ineffective treatment with potential adverse effects in the nonresponders. We demonstrate that noninvasive PET/CT imaging of CD69 may allow early detection of immunotherapy responsiveness in patients with GBM.


Introduction
Glioblastoma (GBM) is the most malignant brain tumor in adults, with a median overall survival (OS) of 14-16 months from initial diagnosis despite aggressive multimodal therapy (1,2). The current standard of care for GBM comprises maximal safe surgical resection followed by concurrent chemo radiotherapy (CCRT), maintenance chemotherapy with temozolomide (3), and tumor-treating fields (TTF; ref. 4). Because of the aggressive and infiltrative nature of GBMs, tumor recurrence is inevitable after initial therapy (5). At recurrence, treatment options are largely palliative and associated with only a partial response and variable survival benefits (6).
To identify an effective treatment for brain tumors, several immunotherapeutic approaches have been introduced to harness the patient's immune response to fight and eliminate tumor cells. Although immune checkpoint inhibitor (ICI)-based immunotherapy has revolutionized the treatment of several cancers including metastatic melanoma (7), non-small cell lung cancer (8), and renal cell carcinoma (9), the objective response rates for these cancers are only up to approximately 50% (10). ICIs are mAbs that block proteins present on T cells from binding to inhibitory ligands present on tumor cell surfaces, thereby preventing tumor cells from suppressing the activity of cytotoxic T cells and resulting in a profound inflammatory response within the tumor bed. The key players in inhibitory checkpoint signaling pathways include programmed cell death protein-1 (PD-1) and cytotoxic T-lymphocyte-associated protein-4 (CTLA-4; ref. 11).
Although progress has been made in the development of immunotherapies, the available imaging tools capable of quantifying and monitoring responses to these therapies remain limited in the clinical field. Clinical MRI is the mainstay for assessing treatment response to therapy in neuro-oncology. However, it exhibits morphologic features and is thus nonspecific (20). For most neuro-oncology treatments, including ICI, patients continue treatment until radiographic evidence of tumor progression by MRI (21,22). Consequently, nonresponding patients who would otherwise have the opportunity to attempt potentially more effective treatment regimens may spend most of their remaining months on ineffective treatment and might experience severe side effects. Hence, there is a need to develop novel and more effective imaging strategies that provide molecular information and determine early on whether a patient responds to immunotherapy.
PET is a unique nuclear medicine modality that enables the real-time imaging quantification of molecular processes by administering a radiolabeled tracer that attaches a high-affinity peptide or protein to a cell surface receptor. The most commonly used tracer is fluorine-18-fluorodeoxyglucose ( 18 F -FDG). FDG-PET is an imaging method based on the increased rate of glucose metabolism (23,24) and is sufficient for monitoring therapeutic effects in most malignancies; however, its role in brain oncology is limited, as it exhibits low specificity due to its accumulation in all hyper metabolic cells, specifically glucose-avid normal brain cells (25).
Recently, several studies have focused on engineering new tracers, using biologics labeled with long half-life radioisotopes such as Zr-89 (78.4 hours; ref. 26) and targeting checkpoint molecules such as PD-1/programmed death-ligand 1 (PD-L1)/cytotoxic T-lymphocyte associated protein 4 (CTLA-4) to predict the response to ICI in murine models (27) and clinical trials (28,29). However, while in a clinical trial evaluating PD-L1 immuno-PET, standardized uptake value (SUV) showed a correlation with response, the signal uptake in tumors was heterogeneous, varying within and among lesions, patients, and tumor types (29). Moreover, emerging varieties of cancer immunotherapies highlight the need for the direct evaluation of immune cells.
PET imaging probes that selectively depict effector T cells, which act as key players in tumor eradication, may be valuable for therapeutic assessment. PET antibodies that target effector immune cells are currently being evaluated in clinical trials. In a previous study (30), patients with metastatic solid tumors undergoing ICI therapy underwent PET imaging with an anti-CD8 radiolabeled minibody and CD8 + tumor-infiltrating lymphocytes (TIL) were detected in tumors 24 hours postinfusion. However, one known limitation of imaging CD8 to examine antitumor immunity is that many CD8 + TILs can be bystander cells, which likely have little effect on the clinical response (31).
Furthermore, a group from Stanford University reported successful developments in immune system visualization techniques using OX40 mAb for imaging OX40 + activated T cells in a mouse model of syngeneic lymphoma (32) and 89 Zr-DFO-ICOS mAb tracer, for detecting ICOS + T cells in a murine lung cancer model (33). Following these promising results, they extended their pursuit to the more challenging type of tumor, GBM, and demonstrated that 89 Zr-DFO-OX40 mAb PET specifically delineates stimulated lymphoid organs following the administration of a cancer vaccine applied in an orthotopic GBM model. However, direct evaluation of intratumoral lymphocytes between responders and nonresponders was unachievable; therefore, they could not corroborate the response resulting from the migrated cytotoxic T cells (34).
Cluster of differentiation 69 (CD69), a C-type lectin protein, is one of the earliest cell surface proteins expressed by activated lymphocytes (35)(36)(37). It is also involved in lymphocyte proliferation and functions as a signal-transmitting receptor (38). CD69 is expressed by mature activated T cells and platelets and is not found in resting circulating leukocytes in humans (37). It is also a marker for tissue-resident memory T-cells (predominantly outside the CNS; ref. 39).
Thus, CD69 is a general surface marker for activated immune cells in tissues with limited presence in the circulation, and its specific functional and fundamental expression on T cells suggests that it could act as a biomarker of activated antitumor T cells. This is further supported by recent work on a murine colon carcinoma model that showed radiolabeled CD69-directed Ab combined with PET imaging as a noninvasive method to assess the early response to ICI with increased uptake in the tumors and lymphoid tissue of ICI-responsive tumor-bearing mice (46).
In this study, we utilized Ab-based PET/CT imaging (immuno-PET) with 89 Zr-DFO-CD69 Ab to achieve noninvasive observations of the immune response in preclinical GBM. We demonstrate the feasibility of early visualization and quantification of T-cell activation in response to ICI immunotherapy in a murine GBM model, with a positive correlation with survival. Together with scRNA-seq data of recurrent GBM tissues from patients receiving versus not receiving ICI, our study shows the potential incorporation of CD69 immuno-PET as a response assessment to immunotherapy in patients with GBM.

CNS and Spleen Tissue Processing
Single-cell suspensions were obtained from spleen and brain tissues, as described previously (47). Briefly, splenocytes were isolated by mechanical disruption of the spleen, passage through a 40-μm filter, and red blood cells (RBC) lysis using ACK lysis buffer (Gibco). Minced tumor tissues were filtered through a 70-μm cell strainer and incubated in a 1X collagenase IV cocktail [1 mL 32 mg/mL (7.4kU) collagenase IV (Worthington, LS004209), 10 mg (53 kU) DNase1 (WorthingtonLS002139), and 20 mg soybean trypsin inhibitor (Worthington LS003587)] for 45 minutes. The samples were then centrifuged for 10 minutes at 2,000 rpm. Cell pellets were suspended in serum-free cell freezing media (Bambanker) at 1 × 10 6 /mL for tumor samples and 3 × 10 7 /mL for spleen samples and were cryopreserved at −80°C until further use.

Cell Lines
GL261 cells were obtained from NCI Division of Cancer Treatment and Diagnosis Tumor Repository. Human Jurkat T cells (clone E6-1) were obtained from ATCC. Cells were maintained in liquid nitrogen in Bambanker cryopreservation media until further use. Cells were tested for Mycoplasma using Lonza MycoAlert Mycoplasma Detection Assay Kit prior to any use. Cells were used within 3-5 passages.

In Vivo Treatments
Following injections, mice were divided into two or three different treatment groups (depending on the specific experiment, as detailed in the Results section): ICI treatment, ICI and CD69 blocking, and a DPBS-injected (vehicle) control group. Two ICI treatments were administered on days −3 and 0 in comparison with the tail vein injection and consisted of 200 μg of each ICI (anti-CTLA-4 and anti-PD-1), whereas the control group was injected with 100 μL DPBS per mouse. All injections were of equal volume and were delivered intraperitoneally. On day −1, 500 μg anti-CD69 was injected intraperitoneally into each mouse in the preloaded unlabeled CD69 Ab + ICI-treated group. The experiment was repeated 2-3 times as detailed in the Results section.

Immunohistology Analysis
GL261 tumors from ICI-treated mice were fixed for 24 hours in neutral phosphate-buffered 10% formalin (Thermo Fisher Scientific) and transferred to 70% ethanol for processing. The tissue samples were dehydrated using a 70% to 100% ethanol gradient over 12 hours, cleared with histologic grade xylene (Leica), embedded in paraffin (Paraplast Plus, Leica), sectioned (4 μm) on Superfrost Plus slides (Thermo Fisher Scientific) using a Leica RM2235 microtome, and baked for 30 or 60 minutes at 60°C. Thereafter, separate staining protocols were used to perform hematoxylin and eosin (H&E) and immunofluorescence (IF) staining.
A Leica ST5020 slide stainer with an integrated cover slipper, CV5030, was used to perform H&E staining with the following staining reagents from Leica: hematoxylin 560MX, blue buffer, aqua define MX, and eosin 515 phloxine. H&E-stained tissue sections were imaged using a Revolve G102 microscope (ECHO) at 1× magnification.
For IF staining, tissue sections were incubated with anti-mouse polyclonal CD69 (AF555, Abcam) and anti-mouse CD3 (AF488, clone 17A2, BioLegend) fluorescence-conjugated antibodies overnight at 4°C. Secondary goat antirabbit antibodies were used to detect primary conjugated anti-CD69 antibodies. Tissue sections were additionally stained with Hoechst 33342 (Cell Signaling Technology) to detect cell nuclei following each primary staining incubation as described previously. The stained tissue sections were imaged using a Nikon Ti2-E Eclipse microscope at 40× magnification. NIS Elements software (version blank) was used to analyze the slides for positive staining of CD69 and CD3 antibodies in the tumor tissue. IF Ab staining was performed as described previously with modifications as required (49).
For IHC staining, slides were deparaffinized and rehydrated using a standard histology protocol. Antigen retrieval was performed using citrate buffer (Cell Signaling Technology). The antibodies used were CD69 from GeneTex (GTX37447), and CD3 from Cell Signaling Technology (99940), which were applied using a 1:300 (CD69) or a 1:100 (CD3) dilution at room temperature. The secondary Ab consisted of a Boost Rabbit HRP Polymer (Biocare Medical). The substrate used was 3,3, Diaminobenzidine (Dako), and the slides were counterstained with hematoxylin (Dako).

MRI
MRI was used to confirm tumor presence and subsequently calculate tumor volume. Mice were anesthetized via a nose cone with 1%-2% isoflurane and O 2 , positioned on an animal bed, and then placed in the scanner. MRI was performed using a 7T/30-cm AVIII spectrometer (Bruker Biospin) equipped with a 12-cm gradient set, an actively decoupled 86-mm quadrature RF volume transmit coil, a 2-channel mouse brain receiver array, and Paravision 6.0

Preparation of 89 Zr-DFO-CD69 Ab
The conjugation and radiolabeling procedures were performed as previously described, with slight modifications (50). For conjugation, a five-molar-fold excess of p-SCN-Bn-DFO (13.3 μL of 5 mmol/L solution) was added to 1 mL of CD69 Ab (2 mg/mL). The pH was adjusted to 9 by using sodium carbonate (0.1 mol/L). The reaction progress was monitored by size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) using an Agilent 1260 Infinity HPLC (Agilent Technologies) equipped with a Bio SEC-3 4.6 mm x 300 mm column (Agilent Technologies). Using Hamblett method, a DFO substitution number of 1.02 ± 0.26 DFO/Ab was determined (51).
For radiolabeling, we used 89 Zr, an ideal radioisotope for Ab-based PET imaging studies, because of the consistency between its half-life (t 1/2 = 78 hours), mAb localization, and clearance rates (52)(53)(54)(55). A total of 130 MBq 89 Zr oxalate was used to radiolabel the DFO-CD69 Ab conjugate (1.17 mg/mL). To assess the progress of 89 Zr labeling, instant thin-layer chromatography was performed using a FlowCount radio-HPLC detection system (Eckert & Ziegler, model 106), then the reaction contents were buffer exchanged to DPBS by PD-10 size exclusion. To determine the radiochemical yield and purity of the conjugated and labeled radiotracers, SEC-HPLC was performed using an Agilent 1260 infinity HPLC (Agilent Technologies) equipped with a Bio SEC-3 4.6 mm × 300 mm column (Agilent Technologies). Specifically, a UV-Vis wavelength of 280 nm was used, which is well-suited for visualizing IgG antibodies (56). First, we ran multiple different IgG antibodies and found that they consistently eluted for approximately 6.7 minutes using an Agilent Bio-SEC 3 column. Successful conjugation without aggregate formation was observed. The gamma radiation elution time of 89 Zr-DFO-CD69 Ab was 8 minutes, a 1.2-minute delay compared with the elution time of its spectral absorbance, due to the tandem detection of spectral absorbance followed by gamma radiation. The gamma radiation of 89 Zr-DFO eluted at approximately 10.9 minutes following the spectral absorption of DFO at 9.7 minutes.

Immunoreactivity
A bead-based radioligand binding assay was performed to assess tracer-binding specificity (57). Recombinant mouse CD69 (R&D Systems) was bound through its Histidine 6-tag to His-tag isolated Ni-NTA Dynabeads (Invitrogen). Nonspecific binding to Ni-NTA was blocked using an IgG Ab with a Histidine-6 tag. These beads were then incubated with 89 Zr-DFO-CD69 Ab, with or without CD69 Ab (InVivoMAb anti-mouse CD69, clone CD69.2.2). As a control for nonspecific bead binding, 89 Zr-DFO-CD69 Ab was incubated with Dynabeads https://doi.org/10.1158/2767-9764.CRC-22-0434 | CANCER RESEARCH COMMUNICATIONS CD69 Immuno-PET for Glioma Immunotherapy and Ni-NTA was blocked with IgG-Histidine-6 Ab. After incubation, the supernatant and beads of each sample were separated and collected to quantify radioactivity in a gamma counter using counts per minute. Triplicates of each experimental condition were used to ensure consistency in the assay results.

PET/CT and Biodistribution Studies
Immediately after radiotracer production, each mouse was injected intravenously with 100 μL of 89 Zr-DFO-CD69 Ab (24-46 μg Ab, 1.4-4 MBq 89 Zr, specific activity: 8.79-13.33 GBq/μmol; a range is depicted since the experiment was repeated). To examine the effect of preloading with unlabeled CD69 Ab, one group was injected intraperitoneally at day −1 in comparison with tail vein injection, with 500 μg of CD69 Ab, reducing the specific activity 10-fold.
Subsequently, static immuno-PET imaging was performed using an Inveon Preclinical Imaging Station (Siemens Medical Solutions) at 1, 2, 3, 4, and 6 days posttreatment and tracer tail vein injection. All images were coregistered using the Vivoquant software (version 9), and ROI analysis was guided by CT.
Tumor-specific ROIs were generated and presented as the maximum or mean SUVs (SUV max or SUV mean ). The ROI was also drawn around the left ventricle of the heart at the apex to calculate the blood pool activity as a measure of nonspecific signals, and the SUV max or SUV mean tumor-to-blood ratio (TBR) was calculated. Biodistribution (BioD) studies were performed immediately after the last imaging (day 6) and on days 3 and 8 for ICI-only treated mice. The major organs were collected and weighed, and tissue-associated radioactivity was assessed using a gamma counter as the percent injected dose/gram (%ID/g).
The biodistributed samples were counted using a Wizard 2-Detector Gamma Counter (Perkin Elmer, model 2480).

Single-Cell RNA Sequencing
Two primary GBM scRNA datasets (GSE84465 and GSE131928) were downloaded from the Gene Expression Omnibus (GEO) database (58,59). One dataset that filtered CD45 + cells from primary, recurrent, and post ICI treatment GBM samples (GSE154795) was downloaded from the GEO database (60). Smart-seq2 data were normalized to transcripts per million (TPM), and the normalization of GSE154795 was the same as in the original study (60).
Quality control, dimensionality reduction, and cell clustering were performed using Seurat v4. The cell types of the different clusters, annotated using cranial markers, were based on the original study. Visualization of the expression and coexpression of CD69 in different cell types was performed using the Seurat FeaturePlot function and ggplot2 package in Uniform Manifold Approximation and Projection (UMAP) for embedding. Statistical significance was evaluated using ANOVA for the three groups, and pairwise comparisons were evaluated using the Wilcoxon rank-sum test for nonnormally distributed datasets. All the single-cell RNA sequencing (scRNA-seq) datasets we tested had P < 2.2e-16 in Shapiro-Wilk test and P < 3.7e-24 in Anderson-Darling normality test, denoting that these samples do not have normal distribution. All analyses were performed using R version 4.1.

Statistical Analysis
Graphics were created using BioRender (https://biorender.com). Statistical analyses, including unpaired Student t test and one-or two-way ANOVA, were performed using GraphPad Prism v9.2.0 software. Where appropriate, statistical significance was corrected for multiple comparisons using Tukey method or, for unequal SE, using Welch correction. Kaplan-Meier survival curves were generated to determine survival and then compared using the log-rank Mantel-Cox test. Pearson correlation test was used to determine the relation-ship between the immuno-PET signals and survival. All data are presented as the mean ± SEM. Statistical significance was determined as indicated in the text and the figure legends. Statistical significance was set at P < 0.05. P values are as follows: NS, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Data Availability Statement
The scRNA-seq datasets are available through GEO (GSE84465 GSE131928, and GSE154795) and from the authors of these data (58)(59)(60).The data analysis generated in this study is available upon request from the corresponding authors.

CD69 is Upregulated Upon T-Cell Activation In Vitro
To corroborate our hypothesis that CD69 is an ideal imaging agent for monitoring ICI response by virtue of early T-cell activation, we measured CD69 expression on T cells following activation in vitro and on GL261 glioma cells.
Healthy mouse splenocytes were activated with anti-CD3/CD28 and IL2, and  Fig. S1E). Taken together, CD69, an early activation marker in mouse and human T cells, demonstrates robust expression at 24 hours postactivation and is not expressed in GL261 glioma cells or nonhematopoietic cells within the GL261 tumor microenvironment (TME); suggesting that it can be a suitable biomarker for immuno-PET imaging.

In Vivo Early Activation of CD69 on TILs Following ICI in a GBM Mouse Model
To 12 after tumor cell inoculation. T-cell populations were examined in dissociated tumors and splenocytes by flow cytometry on days 0 (pretreatment), 2, 4, and 6, relative to treatment (summarized in Fig. 1A). In the ICI-treated group, the proportion of CD8 + TILs significantly increased over time while the proportion of CD4 + TILs decreased, when compared with pretreatment. Similar trends in Tcell populations were observed in the control group, although not significant (Fig. 1B). We also quantified the absolute number of TILs per cubic-millimeter (mm 3 ) of tumor (cell density in tissue) to represent the amount of T-cell accumulation. In the control group, no increase in TILs was observed compared with pretreatment. However, in the ICI-treated group, the number of CD8 + TILs increased following ICI treatment (Fig. 1C). CD4 + TILs showed a similar trend as CD8 + TILs, but was not significant (Fig. 1C). The distribution of Tcell populations in the spleen in the ICI-treated group demonstrated an overall trend of decreased CD8 + and increased CD4 + T cells over time, which reached statistical significance when compared with pretreatment. This trend was not found in the control group, where CD4 + T cells kept an overall stable percentage over time, and CD8 + T cells showed a trend of increase in proportion over time that did not reach any significance (Fig. 1D). Overall, these data demonstrate elevated CD8 + TILs and their decreased proportions in the periphery upon ICI treatment (Fig. 1B-D). These findings are in line with previous reports of high intratumoral, but not circulating, CD8 + T cells as predictors of ICI treatment outcomes (66). We then investigated the kinetics of CD69 expression on TILs and splenic T cells after ICI treatment in relation to PD-1 expression, a marker previously evaluated as an immuno-PET target on TILs (ref. 67; Fig. 2). CD69 + expression was significantly increased on CD8 + and CD4 + T cells both within the tumor ( Fig. 2A) and spleen (Fig. 2B). Notably, a significant increase in the expression of CD69 + on TILs was observed at an earlier timepoint (2 days) compared with that in splenic T cells (4 days). Moreover, on day 2, there was a significant difference between CD69 expression on TILs and splenic T cells, as shown by the ratio of post/pretreatment CD69 + /CD8 + T cells (Fig. 2C). Similar kinetics of PD-1 and CD69 were observed in splenic T cells (a significant increase in 4 days; Fig. 2B and E); however, upregulation of PD-1 + on TILs occurred at a later timepoint (6 days; Fig. 2D and F), compared with CD69 (2 days; Fig. 2A and C). As with CD69, PD-1 expression on TILs at the mentioned timepoint (6 days) was significantly higher than that on splenic T cells (Fig. 2F). Together, these data demonstrate that CD69 is an early T-cell activation marker specifically in the TME after ICl, demonstrating its potential for early immuno-PET monitoring of ICI response.

CD69 within GBM is Predominantly Expressed on Lymphocytes and Increases Following ICI Treatment in a Mouse Model and in Patients
To further validate the expression of CD69 on TILs, we visualized orthotopic GL261 tumors from ICI-treated mice using microscopy following H&E, IF, and IHC staining.  Fig. S2C and S2D, respectively). In addition, we performed IHC staining to evaluate the expression of CD69 on TILs from GL261 tumorbearing mice in control and ICI-treated animals. As positive control staining, we evaluated IHC on spleen tissue for CD3 and CD69 (Supplementary Fig.   S2E). In the ICI-treated group, TILs were identified in different areas with CD3 expression, and CD69 staining on serial section slides suggested coexpression of CD69 on TILs (Supplementary Fig. S2F). Control mice exhibited clear CD3 staining without corresponding CD69 expression (CD69 had background nonspecific staining; Supplementary Fig. S2G). Together, these data support the expression of CD69 on CD3 + T cells in the tumor and spleen tissues of GBMbearing mice following ICI treatment and further support its use as a putative biomarker of immunotherapy response.
Next, we examined the potential of CD69 as a biomarker of ICI response in patients with GBM. scRNA-seq data were analyzed from two published datasets of primary IDH-wild-type GBM (58,59). The first dataset comprised a cohort of four primary GBM adult patients (3,589 cells in total), and the other combined scRNA-seq of 20 adults and 8 pediatric patients with GBM (24,131 cells in total). Fig. S3A and S3B). This is in line with our data showing no CD69 expression on GL261 cells or nonhematopoietic cells from dissociated GL261 tumors ( Supplementary Fig. S1). We then examined different immune cell types from scRNA-seq data and found that CD69 expression was significantly higher on T cells than macrophages ( Supplementary  Fig. S3C), supporting the notion that CD69 is a prominent marker of T-cell activation.

We first examined CD69 expression across different cell types and found that it was expressed almost exclusively on immune cells and not on tumor cells or CNS resident cells (Supplementary
We next examined the influence of ICI treatment on CD69 expression, and the ability of this molecule to serve as an immunotherapy response marker relevant to patient imaging. To do this, we used a scRNA-seq dataset that examined the impact of neoadjuvant anti-PD-1 (neo-aPD-1) on the immune landscape of the TME. The dataset included tumor-infiltrating CD45 + immune cells isolated from 40 patients: 14 newly diagnosed GBM (GBM.new), 12 recurrent GBM without prior immunotherapy (GBM.rec), and 14 recurrent GBM with neo-aPD-1 therapy (GBM.pembro; n = 156,766 cells in total; ref. 60). CD69 was predominantly expressed on CD3 + lymphocytes and, to a lesser extent, on myeloid/microglia cells, with an apparent increase following neo-aPD-1 treatment ( Fig. 3A and B; Supplementary Fig. S3D). Analysis of the scRNA-seq lymphocyte cluster showed the highest amounts of CD69, CD3, CD8 in pembro.GBM compared with the "new" and "recurrent" GBM groups (Fig. 3C). Also, the pembro.GBM group had the highest expression of granzyme-B (GZMB) and PD-1 (PDCD) genes and lowest expression of CD4 and TIM3 (HAVCR) genes (Fig. 3C). We further investigated T-cell subpopulations from the scRNA-seq data and found significant increase of CD69 expression across cell types including on early activated T cells, effector CD8 + , and effector progenitor T cells in the cohort of GBM.pembro compared with the other two cohorts (Supplementary Fig. S3E). These T-cell subsets are known to contribute to antitumor immunity (73)(74)(75). Of note, CD69 expression was increased following ICI treatment on T-regulatory cells, but only when compared to GBM.new patients but not GBM.rec (Supplementary Fig. S3E). Together, these data support that CD69 is a putative biomarker for monitoring changes of T-cell immune responses in the TME following ICI administration in patients with GBM.
To evaluate 89 Zr-DFO-CD69 Ab for in vivo immuno-PET, mice with established GL261 tumors treated with ICI or vehicle control received tail vein injection of 89 Zr-DFO-CD69 Ab. Subsequently, static PET/CT imaging was performed 1, 2, 3, 4, and 6 days posttreatment and tracer injection. BioD analysis was performed after the final immuno-PET scan on day 6 (summarized in Fig. 4A).
We performed a semiquantitative analysis of the tracer signal by determining the SUV max of the tumor-specific regions. At every time point assessed, immuno-PET scans showed a significantly higher SUV max in the tumors of ICI-treated mice than in the control group. Furthermore, in each group, CD69 expression increased over time ( Fig. 4B and C). Notably, the most significant difference between the groups was observed on day 2. These data confirm that CD69, a marker for T-cell activation, can be detected by immuno-PET and is significantly higher after ICI treatment, with an increase in tracer SUV over time.
Next, specific 89 Zr-DFO-CD69 Ab PET imaging uptake was calculated by dividing the tumor uptake by left heart ventricle uptake to derive the TBR, a previously reported measure of tumor-retained signals (81). An SUV max TBR >1 putatively represents refined tumor-specific uptake that controls nonspecific tracer signals from the blood. Tumor-specific tracer uptake was significantly higher in the ICI-treated group than in the control group at all time points. In addition, in the ICI-treated group, tumor-specific uptake was observed starting on day 2 and increased over time. Strikingly, on day 6, we noted very specific tumor uptake, as indicated by SUV max TBR >2. However, in the control group, tumor-specific uptake was noted only on day 6 and was only slightly higher than that in the blood (SUV max TBR mean = 1.27; Fig. 4D). Evaluation  Fig. S6A).
Notably, SUV max values generated following manual tumor segmentation provide higher interobserver precision than SUV mean values as the former is not influenced by mild differences in segmentation. These results suggest that our tracer uptake depicts a unique ICI-induced immune process occurring at the tumor site and not only a representation of the blood-containing tracer within the tumor due to a disturbance of the blood-brain barrier.
On day 6, the maximum intensity projection (MIP) 3D method was used to visualize the distribution of the tracer throughout the body. In ICI-treated mice, we observed higher MIP SUV signals at the tumor site compared with the systemic tracer localization found in the chest cavity and upper gastrointestinal system. In our control group, we detected a slightly higher MIP SUV signal at the same general systemic locations, but a much weaker MIP signal at the tumor site (Fig. 4E). These results support that the transition of the tumor from "cold" to "hot" is acquired through increased immune cell activation that is tumor specific as a result of the ICI treatment and not solely a derivative of the blood-contained tracer.
BioD studies performed immediately after the last imaging (day 6) were used to evaluate tissue-associated radioactivity as %ID/g. A significantly higher accumulation of our tracer was found in the tumors of the ICI-treated group than in those of the control group, whereas radioactivity levels in the blood were quite similar between the groups (Fig. 4F). This validates our immuno-PET SUV imaging results and further supports tumor-specific tracer uptake of 89 Zr-DFO-CD69 Ab following ICI treatment. Tracer accumulation in the spleen and thymus appeared to be higher in the ICI-treated group; however, these differences were not significant. No significant differences were observed in other organs ( Supplementary Fig. S6E).
AACRJournals.org Cancer Res Commun; 3(7) July 2023  To further validate the tumor accumulation of our tracer over time, we examined the BioD of 89 Zr-DFO-CD69 Ab on days 3 and 8 after ICI treatment ( Supplementary Fig. S6B). We observed a significant decrease in the tracer in the blood over time, simultaneously with a significant increase in tracer uptake at the tumor site ( Supplementary Fig. S6C). These data suggest tumor-specific uptake of the tracer, which is not derived solely from the blood tracer content. In addition, we observed an increase in the tracer in the spleen, which is known to have a high lymphocyte content, further supporting the T-cell-specific uptake of the tracer (Supplementary Fig. S6C). Competitive inhibition with CD69 Ab eliminated tracer binding to CD69 in vitro, demonstrating the high specificity of 89 Zr-DFO-CD69 Ab (Supplementary Fig. S4C). In vivo, preloading with unlabeled CD69 Ab administered 24 hours prior to tracer injection significantly reduced tracer uptake in the immune periphery (i.e., spleen) while enhanced tumor-specific 89 Zr-DFO-CD69 Ab uptake, as indicated by the SUV values ( Supplementary Fig. S6D) and BioD data ( Supplementary Fig. S6E). This is in accordance with literature showing that preloading with unlabeled antibodies can improve site-specific PET/CT imaging by competitively inhibiting systemic tracer uptake (82).
Of note, when evaluating an immuno-PET agent with antigen expressed in tissues other than the tumor, on-target off-site "antigen sink" captures radio-tracer resulting in decreased tumor accumulations (83,84). Therefore, in vivo, an isotype control immuno-PET radiotracer will circulate in the blood without getting captured and have noncomparable readouts for our CD69 radiotracer. Thus, a 89 Zr-labeled isotype-matched antibody serves as a poor control for nonreceptor-mediated target tissue accumulation. In addition, to determine the potential for Fc region-mediated radiotracer accumulation, we examined nonspecific staining of in vitro-activated T cells and immune cells from dissociated tumor tissue following ICI treatment and did not detect binding of isotype control antibody, with the MFI of the isotype controls being similar to the unstained cells ( Supplementary Fig. S7A and S7B). These data suggest that the accumulation within the TME is not Fc mediated.

Immuno-PET of 89 Zr-DFO-CD69 Ab as a Prognostic Predictor after ICI Treatment in a GBM Mouse Model
Our data identified CD69 as an immuno-PET biomarker of T-cell activation upon ICI immunotherapy in a GBM model. Because T-cell activation, measured by CD69 upregulation, is key for successful immunotherapy, durable response, and favorable outcome, we tested whether immuno-PET of 89 Zr-DFO CD69 Ab correlates with survival after ICI treatment in our model (summarized in Fig. 5A). MRI data showed that the overall tumor volume and volume distribution were equivalent between the groups the day prior to tracer injection. A slightly higher tumor volume, albeit nonsignificant, was observed in the ICI-treated group, with a mean of 5.74 mm 3 versus 4.86 mm 3 in the control group (Supplementary Fig. S8). The overall survival (OS) was significantly higher in the ICI-treated group, with a mean of 28 days posttumor inoculation compared with 23.2 days in the control group, with HR showing a 35% decrease in mortality in those treated (Fig. 5B).
Correlation analysis ( Fig. 5C; Supplementary Fig. S9) between SUV measurements (SUV max , SUV mean , SUV max TBR, and SUV mean TBR) and survival (i.e., prognosis) with ICI immunotherapy showed a strong positive correlation (r > 0.7) across all immuno-PET time points; higher SUV values, representing T-cell activity, matched longer OS. Even though SUV mean and SUV mean TBRs showed better and more striking results at most time points compared with our SUV max analysis, we prefer to address the SUV max and SUV max TBR results, as we have noted before, as they are more consistent and provide higher interobserver reliability. Hence, when focusing on SUV max analysis, a notable statistically significant correlation was observed on SUV max TBR day 2 after ICI treatment (r = 0.9425, P = 0.016).
While all SUV measurements correlated with the clinical outcome of mice in both groups, we observed inverted results (i.e., a negative correlation) between the SUV measurements and OS in the control group (not treated with ICI), reflecting that higher SUV values correlated with a worse prognosis. It is important to note that the negative correlation was weaker than that in the ICI group, reflected by frequent results of r < −0.5 and a wider range (r = −0.15 to −0.99). As in the ICI group, the most notable statistically significant correlation in the control group was observed in SUV max TBR; however, this occurred on day 3 (r = 0.9937, P = 0.0063) compared with day 2 in the treated group ( Fig. 5C; Supplementary Fig. S9).
To investigate the role and strength of our functional CD69 immuno-PET imaging, we assessed anatomic information obtained by longitudinal MRI at the same time frame as the immuno-PET in both ICI treatment and control group. Unlike the statistically significant changes observed from immuno-PET CD69 signal, tumor sizes obtained by MRI were not significantly different between the groups at these timepoints, despite some mild reduction in tumor size for the ICI-treated group on day 6 ( Supplementary Fig. S10).
These data show that immuno-PET of 89 Zr-DFO-anti-CD69 not only correlates with prognosis upon ICI immunotherapy and is therefore a potential imaging tool for immunotherapy treatment guidance but may also serve as an imaging tool for prognosis prediction in nontreated patients, especially when using SUV max TBR measurements on days 2 and 3, respectively.

Discussion
GBM is an aggressive brain tumor with limited therapeutic options (1, 2), which has motivated the investigation of more effective therapies. Immunotherapy represents a significant advance in the treatment of many cancers and may hold promise for GBM treatment. However, oncologists must balance the benefits that are eventually observed in a small group of patients with immune-related adverse events (85).
Standard clinical imaging modalities, typically used to monitor therapeutic efficacy, have shown limited success in immunotherapy. This is partly due to the apparent radiologic tumor progression from treatment-induced inflammatory infiltrates (86), which is difficult to distinguish from true tumor progression (87,88). Therapeutic response assessments are currently based on presumed tumor progression rather than on earlier predictive biomarkers of response to therapy. Therefore, alternative imaging methods and biomarkers for monitoring early antitumor immune responses are urgently needed. To date, several successful methods for visualizing specific immune markers have been developed for systemic cancers (33,(89)(90)(91), with limited success for GBM, posing greater challenges for immuno-PET monitoring (34). In this study, we investigated the utility of CD69 immuno-PET for molecular imaging to predict therapeutic responses to immunotherapy for GBM.
CD69 is a transmembrane protein that is predominantly expressed on T cells, with some expression on other immune cells, including NK cells (35,38,92). CD69 is rapidly upregulated on T cells upon productive T-cell receptor (TCR) engagement with cognate antigens (62), and thus can be a promising biomarker of T-cell activation for early immunotherapy response monitoring. Recent work on CD69 immuno-PET in a murine colon carcinoma model demonstrated an association between tracer uptake and response to ICI (46), further supporting the use of CD69 immuno-PET for immunotherapy response assessment in GBM.
Here, we demonstrate that CD69 can be detected by PET in the GBM TME to monitor ICI treatment response. Our immuno-PET of 89 Zr-DFO-CD69 Ab in a murine GBM model enabled repeated, quantitative, and noninvasive PET imaging assessments. We found significantly higher tracer uptake in the tumor area of ICI-treated GBM mice than in vehicle-treated mice. In addition, and important for clinical translation, we showed a strong positive correlation between SUV and survival after ICI immunotherapy. Interestingly, our data on high TBR using PET and BioD demonstrated a specific immune activation process occurring within the tumor site.
Our in vitro and in vivo studies consistently pointed to 48 hours post ICI treatment as the time point for high CD69 signal expression, and a strong association with survival. If future studies show similar findings, this early time point could have important implications for clinical practice. For instance, patients can be administered intravenous with both ICI and the tracer on the same day and then back for only one immuno-PET imaging two days later, allowing the process to be efficient for the early detection of responses.
While we observed a strong positive correlation between survival and SUV in the ICI group, we observed a negative correlation between SUV measurements and OS in the control group (higher SUV values correlated with worse prognosis). These data show that immuno-PET of 89 Zr-DFO-CD69 Ab may also serve as an imaging tool for prognosis prediction in patients outside the context of immunotherapy. Interestingly, tumor sizes from MRI data on the day prior to tracer injection, between doses on ICI, did not correlate with either survival or CD69 SUV signals in the ICI-treated group, and MRI on day 6 could not provide evidence of difference between the treatment groups. Overall, these data suggest that CD69 immuno-PET is able to detect changes in the tumor immunobiology prior to changes in tumor size, strengthening its potentially useful as a predictive biomarker of ICI responses.
Consistent with our mouse data, our analysis of scRNA-seq data obtained from specimens of patients with recurrent GBM treated with neoadjuvant anti-PD-1 and control cohorts revealed that CD69 expression, along with other effector markers, was significantly elevated on TILs from ICI-treated patients compared with the control group. This finding strengthens the rationale for its translation into clinical practice. We hope, future studies would assess the utility of CD69 immuno-PET in predicting responses in clinical trials and routine clinical work to distinguish treated responders from non-responders.
Non-responders could avoid the often-severe side effects of ineffective costly therapy and would have an opportunity to try other, potentially more effective regimens, immunotherapy-based, or otherwise, at an earlier time point.
This study has several limitations. In this study, we used the GL261 model to assess our tracer for monitoring immunotherapy responses in GBM. While GL261 has major benefits as a well-established orthotopic syngeneic model, which allows for evaluation of immunotherapies (93,94), one of the limitations of GL261 is that its high mutational burden may not represent the majority of patients with GBM (94,95). Future studies validating CD69 immuno-PET in genetically engineered or de novo mouse GBM models can therefore improve relevance for a heterogenous population of patients with GBM. In addition, while our study was performed under "sterile" conditions, in which no external or internal immune challenges are introduced besides immunotherapy, patients usually receive ICI following or in parallel to other treatments. One example is corticosteroids, which are often prescribed to decrease local edema and suppress the immune response, which might affect the immuno-PET results (96).
Another example is intercurrent infection, which is commonly introduced during the disease process, and its effects on immune activation. Therefore, future studies should evaluate the effects of "real-world" events on imaging. In addition, we assessed CD69 immuno-PET only for short-term ICI immunotherapy; however, this strategy may be informative for monitoring long-term ICI treatment and other T cell-mediated immunotherapies. Therefore, the validation of CD69 immuno-PET imaging for these scenarios is warranted in future studies. Finally, this study did not assess the biologic impact of antibody binding to CD69, which although given in small doses, may influence T-cell activity. Previous work has shown that anti-CD69 Ab can enhance T-cell antitumor activity in systemic cancers (68,77,78). While future studies should assess the dose effects of anti-CD69 administration in GBM, we anticipate that optimization of molar activity can reduce the amount of the anti-CD69 Ab that would be administered.
Overall, we demonstrated that CD69 on TILs represents a promising biomarker for early detection of response to ICI therapy in GBM. Preclinical CD69 immuno-PET using 89 Zr-DFO-CD69 Ab was found to be highly sensitive for the detection of the anti-GBM immune response and served as a predictor of prognosis upon ICI therapy. Furthermore, we showed that CD69 expression is upregulated also in scRNA-seq obtained from ICI-treated patients, thus providing a strong translational rational. Given the relatively short survival times of patients with GBM and the increasing number of immunotherapies being evaluated, early measurement of therapeutic efficacy can have drastic implications for clinical care, preventing unnecessary side effects of ineffective therapies, allowing patients more time to receive alternative therapeutic regimens, and providing validity for continuing treatment in responding patients. We envision clinically, patients may serve as their own baseline, and pretreatment PET scan may serve as appropriate baseline to assess the increase in PET signal upon ICI. Overall, given our promising results, we believe that CD69 immuno-PET imaging should be further developed for clinical evaluation and tested in other immunotherapies to assess its ability to be used as a general assessment tool for immunotherapy response.